This competitive renewal requests continued support for our investigation on integrin signaling, a central event for many cell adhesion-dependent physiological and pathological responses. Integrins are a class of heterodimeric (a/b) transmembrane receptors that are activated via a distinct mechanism, i.e., upon cellular stimulation, a latent integrin can receive intracellular signal(s) at the cytoplasmic face, which is transmitted via the transmembrane region to the extracellular domain, converting it from a low to a high affinity ligand binding state. Since early 1990s, this so-called integrin "inside-out" signaling or integrin activation has been under intensive studies as evidenced by nearly 12,000 articles in PubMed. In late 1990s, the field gained significant momentum due to the discovery of the actin linking protein talin as an integrin activator. We proposed, based on NMR and mutagenesis data that talin activates integrin by disrupting a key integrin cytoplasmic clasp and triggering the long-range inside-out conformational change of the receptor. This model has been validated by extensive follow-up studies and is now widely accepted. However, major knowledge gaps remain. In particular, little is known about how integrin is dynamically regulated, and what factor(s) controls the balance between the resting and activating states of the receptor. The issues are fundamental to our understanding of integrin- mediated cellular events such as cell spreading and migration. They are also of high medical relevance since uncontrolled integrin activation is known to be directly linked to human disorders such as thrombosis, stroke, and cancer. This proposal will focus on investigating an emerging regulatory machinery, which involves filamin as a "brake" and migfilin as a "booster" for integrin activation. Specific Aim1 will elucidate the negative regulation of integrin activation by filamin. Specific aim2 will probe the mechanisms of positive regulation of integrin activation by migfilin-kindlin pathway. A suite of structural and functional tools will be used to examine a central hypothesis that integrin is controlled by both positive and negative pathways so as to allow dynamic regulation of integrin signaling, cell spreading, and migration. Our study may lead to a new paradigm for understanding how integrin function is temporally controlled. The results may also impact on the therapy for human diseases associated with the dysfunctions of integrin signaling. PUBLIC HEALTH RELEVANCE: The adhesion between cell surface receptor integrin and extracellular matrix (ECM) plays a central role in diverse physiological processes such as embryogenesis, haemostasis, the immune response and the maintenance of tissue integrity. Uncontrolled integrin activation (ECM-integrin interaction) has been linked to a spectrum of human diseases including thrombosis, stroke, atherosclerosis, and cancer. Our proposal will elucidate novel regulatory mechanisms of the integrin activation, which may not only help to understand the fundamentals of cell adhesion but also impact on integrin-related therapy of human diseases such as stroke and cancer.